Nuclear transport of H1 histones meets the criteria of a nuclear localization signal—mediated process

We have studied the nuclear transport of H1 histones using the digitonin permeabilization assay system in order to establish the transport requirements for H1 translocation to the nucleus. Using HeLa cells and fluorescence-labeled calf thymus H1, we show that the H1 nuclear transport in permeabilized cells requires the addition of cytoplasmic extract. Furthermore, it can be blocked by energy depletion and by chilling or by addition of wheat germ agglutinin or by nonhydrolyzable GTP analogs. Thus, the import of H1 histones follows the criteria established for nuclear import mediated by nuclear localization signals (NLS). The distribution of basic amino acids in average H1 sequences, however, does not allow the assignment of a specific element as a classical NLS. J. Cell. Biochem. 64:573–578. © 1997 Wiley-Liss, Inc.     

Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG “swaps” demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.     

Structural basis for the high-affinity binding of nucleoporin Nup1p to the Saccharomyces cerevisiae importin-beta homologue, Kap95p

Macromolecules are transported across the nuclear envelope most frequently by karyopherin/importin-beta superfamily members that are constructed from HEAT repeats. Transport of Kap95p (yeast importin-beta), the principal carrier for protein import, through nuclear pore complexes is facilitated by interactions with nucleoporins containing FG repeats. However, Nup1p interacts more strongly with Kap95p than other FG-nucleoporins. To establish the basis of this increased affinity, we determined the structure of Kap95p complexed with Nup1p residues 963-1076 that contain the high-affinity Kap95p binding site. Nup1p binds Kap95p at three sites between the outer A-helices of HEAT repeats 5, 6, 7 and 8. At each site, phenylalanine residues from Nup1p are buried in hydrophobic depressions between adjacent HEAT repeats. Although the Nup1p and generic FG-nucleoporin binding sites on Kap95p overlap, Nup1p binding differs markedly and has contributions from additional hydrophobic residues, together with interactions generated by the intimate contact of the linker between Nup1 residues 977-987 with Kap95p. The length and composition of this linker is crucial and suggests how differences in affinity for Kap95p both between and within FG-nucleoporins arise.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

Nuclear import of Ran is mediated by the transport factor NTF2

A concentration gradient of the GTP-bound form of the GTPase Ran across nuclear pores is essential for the transport of many proteins and nucleic acids between the nuclear and cytoplasmic compartments of eukaryotic cells [1], [2], [3] and [4]. The mechanisms responsible for the dynamics and maintenance of this Ran gradient have been unclear. We now show that Ran shuttles between the nucleosol and cytosol, and that cytosolic Ran accumulates rapidly in the nucleus in a saturable manner that is dependent on temperature and on the guanine-nucleotide exchange factor RCC1. Nuclear import in digitonin-permeabilized cells in the absence of added factors was minimal. The addition of energy and nuclear transport factor 2 (NTF2) [5] was sufficient for the accumulation of Ran in the nucleus. An NTF2 mutant that cannot bind Ran [6] was unable to facilitate Ran import. A GTP-bound form of a Ran mutant that cannot bind NTF2 was not a substrate for import. A dominant-negative importin-β mutant inhibited nuclear import of Ran, whereas addition of transportin, which accumulates in the nucleus, enhanced NTF2-dependent Ran import. We conclude that NTF2 functions as a transport receptor for Ran, permitting rapid entry into the nucleus where GTP-GDP exchange mediated by RCC1 [7] converts Ran into its GTP-bound state. The Ran–GTP can associate with nuclear Ran-binding proteins, thereby creating a Ran gradient across nuclear pores.     

Evidence for binding of at least two factors, including T-rich strand-binding factor(s) to the single-stranded ARS1 sequence in Saccharomyces cerevisiae.

Summary To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors interacting with the ARS1 region located near the centromere of chromosome IV in Saccharomyces cerevisiae. Using the gel shift assay, we found protein factor(s) which specifically bound to the T-rich strand of the region containing the core consensus and its flanking sequences in ARS1, but not to the opposite strand. We designated this factor ATS (ARS1, T-rich strand-binding factor(s)). Similar specific complexes were also detected with oligonucleotide probes specific for the H4 or C2G1 ARS. As we have previously identified another binding factor, we conclude that at least two factors bind to the single-stranded ARS1 sequence.     

Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p,Nup59p,and Nup170p

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.     

Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export

Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.     

DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

Nuclear import of Pin1 is mediated by a novel sequence in the PPIase domain

Pin1 actively regulates diverse biological/pathological processes, but little is known about the regulatory mechanisms of its cellular localization. In this study, we report that the endogenous Pin1 is distributed in both nucleus and cytoplasm. We found that point mutations of several basic amino acids in the PPIase domain of Pin1 significantly compromise its nuclear localization. Such inhibition is independent of Pin1 enzymatic activity, and is mainly due to the defects in the nuclear import. A novel sequence harboring these residues was identified as a putative nuclear localization signal (NLS) of Pin1. Importin α5 of the nuclear import machinery was found to interact with Pin1.

Structured summary:

MINT-6803320: PIN1 (uniprotkb:Q13255) and importin alpha 5 (uniprotkb:P52294) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)MINT-6803333: importin alpha 3 (uniprotkb:O00505) and PIN1 (uniprotkb:Q13255) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)MINT-6803357: PIN1 (uniprotkb:Q13255) physically interacts (MI:0218) with importin alpha 5 (uniprotkb:P52294) by anti bait coimmunoprecipitation (MI:0006)MINT-6803345: St3 (uniprotkb:P40763) and importin alpha 5 (uniprotkb:P52294) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)     

Single HIV-1 Imaging Reveals Progression of Infection through CA-Dependent Steps of Docking at the Nuclear Pore,Uncoating, and Nuclear Transport

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Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca²⁺ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca²⁺ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M₁ receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca²⁺ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.